In article <cliff.11.3042B554 at molbiol.uct.ac.za>, cliff at molbiol.uct.ac.za wrote:
> Hi all
>> I have an E.coli mutant which was created by TnPhoA mutagenesis. The
> transposon jumped into the 3-phosphoglycerate kinase (pgk) gene in the
>> gapB - pgk - fda
>> glycolytic operon. My question is - does this mean that the fda gene
> downstream of pgk is automatically inactive (polar mutation) OR does
> TnphoA (or Tn5 for that matter) have a known endogenous promoter in its
> inverted repeats ie. will the fda gene be transcribed from within the
> transposon ? The gene would have to be transcribed from within the right
> inverted repeat.
>> The literature (that I have read thus far) is not very helpful in this
> regard.
>> Look forward to hearing from you
> Cliff Dominy
Hello Cliff,
TnphoA does cause polar mutations preventing downstream transcription.
This is the case with every transposon that I have worked with. (TN5,
TN5Lac, TNphoA, Minu-MuLux). You can verify that mRNA transcription is
terminated by the transposon using a fda(DNA) probe against a Northern
blot of mutated vs unmutated E. coli RNA. Your pgk:TNphoA mutant should
not (under normal circumstances) be making any RNA that can bind to fda
specific DNA. Hope this helps.
--
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